In the elimination of cancer cells, virus-infected cells and the like by the living body, cellular immunity, particularly cytotoxic T cells (hereinafter referred to as CTL) play an important role. CTL recognizes the complex formed on cancer cells by a cancer antigen protein-derived antigen peptide (cancer antigen peptide) and MHC (Major Histocompatibility Complex) class I antigen (in the case of humans, referred to as HLA antigen), and attacks/destroys the cancer cells.
As a representative example of the cancer antigen protein, those described in the table of Immunity, Vol. 10, page 281, 1999 can be mentioned. Specifically, gp100 and MART-1, which are melanocyte tissue specific proteins, and melanosome proteins such as tyrosinase can be mentioned. As the cancer antigen protein other than melanoma, cancer markers such as CEA, PSA, and HER2/neu can be mentioned. TERT, whose expression increases in cancer, and the like can also be mentioned. Furthermore, WT1, which is highly expressed in many types of cancer, is also known as a novel cancer antigen protein in leukemia and solid cancers.
The cancer antigen peptide is a peptide resulting from processing of a cancer antigen protein by intracellular protease. As stated above, a complex of this resulting cancer antigen peptide and MHC class I antigen (HLA antigen) is presented to the cell surface and recognized by CTL. However, in developing a cancer immunotherapeutic agent (cancer vaccine) utilizing cancer cell destruction by CTL, it has been difficult to efficiently induce CTL using a cancer antigen peptide alone, because of the generally low immunogenicity. Therefore, there is a demand for the development of a preparation capable of stimulating CTL induction.
To this end, many investigations have been conducted regarding preparations that stimulate CTL induction. Particularly, many investigations have been conducted regarding emulsion type preparations.
Generally, there are different types of emulsion preparations: O/W emulsions and W/O emulsions.
In the case of O/W emulsions, the peptide that serves as the antigen cannot be retained in the inner phase, and therapeutically effective specific CTL induction activity is not exhibited.
In the case of W/O emulsions, in which the water phase serves as the inner phase, excellent CTL induction activity is expected because the peptide that serves as the antigen is easy to retain in the inner phase, but there are many problems to be solved before they are brought into practical applications.
For example, as a W/O emulsion for cancer antigen peptide carrier, incomplete Freund's adjuvant is known, but effective activity cannot be obtained in some cases due to insufficient stability. There have been other problems such as difficulty in administration due to high viscosity.
Incomplete Freund's adjuvant has long been known as an immunopotentiating adjuvant that causes inactivated bacteria or inactivated virus to be included to induce antibody production, and alternative W/O emulsion type vaccine compositions were investigated; for example, the compositions described in Japanese Patent Kohyo publication No. HEI-7-509733, the pamphlet for International Patent Publication No. 94/20071, and Japanese Patent Kokai Publication No. HEI-9-268130 were proposed.
As a substitute for incomplete Freund's adjuvant, an application of W/O/W composite emulsion to vaccine is proposed in Japanese Patent Kokai Publication No. 2001-131087.
However, as a cancer antigen peptide carrier to substitute for incomplete Freund's adjuvant, no W/O emulsion capable of therapeutically effectively activating CTL induction is known.
Generally, because cancer antigen peptides are low-molecular compounds, it is not easy to keep them stable in W/O emulsion. Furthermore, it is feared that the cancer antigen peptide undergoes degradation, aggregation and the like due to the energy during emulsification. Furthermore, cancer antigen peptides range from highly water-soluble ones to slightly soluble ones depending on the amino acid sequence thereof. Therefore, there is a demand for the development of a cancer vaccine based on a highly versatile W/O emulsion that therapeutically effectively stimulates CTL induction, irrespective of the properties of the cancer antigen peptide.
An object of the present invention is to provide an emulsified composition for dilution for preparing a cancer vaccine composition that exhibits effective CTL induction activity in vivo in various cancer antigen peptides, and is of low viscosity and excellent stability. Another object of the present invention is to provide a method of preparing a novel cancer vaccine that specifically stimulates CTL induction in vivo according to the cancer antigen peptide contained, using the above-described emulsified composition for dilution, and the composition.